The Antibiotic Resistance Crisis, with a Focus on the United States

The Journal of Antibiotics (2017) 70, 520–526 & 2017 Japan Antibiotics Research Association All rights reserved 0021-8820/17 www.nature.com/ja

REVIEW ARTICLE

The antibiotic resistance crisis, with a focus on the United States

Evan Martens1 and Arnold L Demain2

Beginning with the discovery of penicillin by Alexander Fleming in the late 1920s, antibiotics have revolutionized the field of medicine. They have saved millions of lives each year, alleviated pain and suffering, and have even been used prophylactically for the prevention of infectious diseases. However, we have now reached a crisis where many antibiotics are no longer effective against even the simplest infections. Such infections often result in an increased number of hospitalizations, more treatment failures and the persistence of drug-resistant pathogens. Of particular concern are organisms such as methicillin-resistant Staphylococcus aureus, Clostridium difficile, multidrug and extensively drug-resistant Mycobacterium tuberculosis, Neisseria gonorrhoeae, carbapenem-resistant Enterobacteriaceae and bacteria that produce extended spectrum β-lactamases, such as Escherichia coli. To make matters worse, there has been a steady decline in the discovery of new and effective antibiotics for a number of reasons. These include increased costs, lack of adequate support from the government, poor returns on investment, regulatory hurdles and pharmaceutical companies that have simply abandoned the antibacterial arena. Instead, many have chosen to focus on developing drugs that will be used on a chronic basis, which will offer a greater profit and more return on investment. Therefore, there is now an urgent need to develop new and useful antibiotics to avoid returning to the ‘pre-antibiotic era’. Some potential opportunities for antibiotic discovery include better economic incentives, genome mining, rational metabolic engineering, combinatorial biosynthesis and further exploration of the earth’s biodiversity. The Journal of Antibiotics (2017) 70, 520–526; doi:10.1038/ja.2017.30; published online 1 March 2017

INTRODUCTION glycopeptides. Antibiotics have been crucial in the increase in life In 1900, infectious disease was the leading cause of death in the expectancy in the United States from 47 years in 1900 to 74 years for world.1 The selective action exerted on pathogenic bacteria and males and to 80 years for females in the year 2000.4 fungi by the ‘wonder drugs’, that is, microbial secondary Over 10 000 microbial secondary metabolites have been metabolites (also known as idiolites), ushered in the antibiotic era discovered.5 The filamentous bacteria, that is, the actinomycetes, are which has been of great importance for human beings ever since. amazingly prolific in the number of antibiotics which they can Antibiotics are low molecular weight compounds, most of which are produce. About 75% of known antibiotics are produced by actino- natural products made by microorganisms or derived from natural mycetes and about 75% of these are made by a single genus, that is, products, which are active at low concentrations against other Streptomyces. Of antibiotics used in medicine, more than microorganisms. Some antibiotics such as sulfa drugs and oxazolidi- 90% originate from the actinomycetes. In a typical actinomycete, nones do not originate from natural products. It is frequently 23–30 gene clusters (about 5% of the genome) are devoted to misrepresented that fluoroquinolones are not of natural product secondary metabolism.6 Also, important are non-filamentous bacteria, origin. Fluoroquinolones are derived from natural products.2,3 such as species of Bacillus, which can produce over 60 antibiotics. The first quinolone was derived from the distillation of quinine Indeed, 12% of known antibiotics are produced by non-filamentous from the bark of the Cincona tree bark. Therefore, the majority of bacteria. In addition, some useful antibiotics, such as fusidic acid, are drugs utilized for chemotherapy against pathogenic microbes are made by fungi (Fusidium coccineum).7 antibiotics. UNDERSTANDING THE ‘CRISIS’ THE ERA OF ANTIBIOTIC DISCOVERY Infectious disease is now the second leading killer in the world, The glorious years of antibiotic discovery, development and produc- number three in developed nations8 and fourth in the United States.9 tion took place in the period between 1940 and the 1960s. Discovery Worldwide, 17 million people die each year from bacterial in later years continued but not as rapidly as in the early years. The infections.10 In the United States, each year approximately two million most important antibiotics include the penicillins, cephalosporins, people are infected with bacteria resistant to antibiotics, of which tetracyclines, aminoglycosides, chloramphenicol, macrolides and 23 000 will subsequently die as a result of these infections.11

1Cempra, Inc., Chapel Hill, NC, USA and 2Research Institute of Scientists Emeriti (R.I.S.E.), Drew University, Madison, NJ, USA Correspondence: E Martens, Cempra, Inc., Chapel Hill, NC 27517, USA. E-mail: [email protected] Received 1 November 2016; revised 29 January 2017; accepted 30 January 2017; published online 1 March 2017 The antibiotic resistance crisis E Martens and AL Demain 521

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for monocytogenes, Mycobacterium bovis, Vibrio cholerae, non-cholera the deaths of 19 000 people and 360 000 hospitalizations in the United Vibrio spp, norovirus, rotavirus, prions, Salmonella spp including States each year12, along with $3–4 billion in US healthcare costs. Salmonella enterica, Shigella spp, Yersinia spp, and toxins from Although MRSA is still a major patient threat, a CDC study published S. aureus, Clostridium perfringens and Bacillus cereus. in the Journal of the American Medical Association Internal Medicine showed that invasive (life-threatening) MRSA infections in healthcare REASONS FOR ANTIBIOTIC RESISTANCE AND HOW TO settings are declining.13 Invasive MRSA infections that began in COMBAT IT hospitals declined 54% between 2005 and 2011, with 30 800 fewer Resistance is enhanced by the use of antibiotics to promote animal severe MRSA infections. In addition, the study also showed 9000 fewer growth and prevent disease in crowded factories and farms. About deaths in hospital patients in 2011 versus 2005.13 30 different antibiotics have been used in food and water for animals. Drug-resistant bacteria kill 25 000 people per year in Europe. At present, more than 50% of antibiotics made are used to promote Perhaps the single biggest public health threat today is antibiotic animal growth. In addition, some developing countries provide resistance.14 An example is gonorrhea, which was treatable by antibiotics without prescriptions, adding to resistance development. penicillin in the 1970s, but is becoming resistant even to ceftriaxone, Antibiotic resistance is due to inactivation by enzymes, such as a third generation oral cephalosporins. Gram-negative infections are β-lactamase, increased efflux of the antibiotic out of cells, decreased becoming untreatable due to resistance elements including extended uptake of the antibiotic, modification of the target to decrease binding spectrum β-lactamases and Klebsiella pneumoniae carbapenemase of the antibiotic, amplification of the target, bypassing the essentiality produced by Enterobacteriaciae. of the target, sequestration of the antibiotic, protection of the target The CDC has categorized the top 18 drug-resistant threats to the and biofilm formation.17,18 A major example of antibiotic resistance is United States based on the specific level of concern: urgent, serious carbapenem-resistant Enterobacteriaceae,19 which causes infections of and concerning.15 Pathogens that are considered urgent threats include the bladder, lung and blood, which can become life-threatening. The Clostridium difficile, carbapenem-resistant Enterobacteriaceae and producing organisms are resistant to almost all antibiotics. They were Neisseria gonorrhoeae. The following pathogens are categorized as discovered in 1998 but the threat did not become recognized until serious threats: multidrug-resistant Acinetobacter; drug-resistant recently. The carbapenems were developed in the 1980s. Resistant Campylobacter; fluconazole-resistant Candida; extended spectrum strains of Klebsiella produce an enzyme called KPC (Klebsiella Enterobacteriaceae; vancomycin-resistant Enterococcus; multidrug- pneumoniae carbapenemase), which breaks down carbapenems. resistant Pseudomonas aeruginosa; drug-resistant non-typhoidal Salmo- Another enzyme destroying the antibiotic is a metallo-beta- nella; drug-resistant Salmonella serotype typhi; drug-resistant Shigella; lactamase. It is found not only in Klebsiella but also in other MRSA; drug-resistant Streptococcus pneumoniae; and drug-resistant enterobacteria such as E. coli. Some of the organisms respond to tuberculosis (TB). Bacteria which are viewed as concerning threats tigecycline and colistin (polymyxin E), but not in all patients. include vancomycin-resistant S. aureus, erythromycin-resistant Group Community-acquired bacterial pneumonia (CABP) caused by A streptococcus, and clindamycin-resistant Group B streptococcus.15 Streptococcus pneumoniae is also becoming a major problem. Resis- Other drug resistance problems include: (1) the intestinal bacterium tance is developing against macrolides such as azithromycin and K. pneumoniae becoming resistant to carbapenems; (2) Escherichia coli clarithromycin. Macrolide resistance has reached nearly 50% in the becoming resistant to fluoroquinolones and causing urinary tract United States. Many strains are not susceptible to pneumococcal infections and (3) Neisseria gonorrhoeae, the bacterium which causes vaccines. New and more potent macrolides are greatly needed.20 gonorrhea, becoming resistant to third-generation cephalosporins. Solithromycin from Cempra, Inc., Chapel Hill, NC, USA, is the only In addition, Clostridium difficile,whichisagrowingproblem macrolide (fourth generation), which has completed clinical develop- in hospitals and long-term care facilities, causes illness in 336 000 ment for community-acquired bacterial pneumonia. The older people in the United States each year and kills 14 000. This represents macrolides inhibit one or two sites on the bacterial ribosome, whereas a 400% increase in both figures from 2000 to 2013. C. difficile is the solithromycin interacts with three distinct sites and is active against leading cause of nosocomial diarrhea around the world and its multi-drug resistant bacteria, including telithromycin-resistant incidence has markedly increased in the period from 2000 to strains.21 2015.16 The infections often occur in the hospital after administration A review on overcoming resistance has been published by the staff of broad-spectrum antibiotics, which deplete the flora of the gut, of The Scientist Journal.22 New strategies that are being explored to allowing endogenous or environmental C. difficile to grow in the combat resistance include: (a) modifying old antibiotics into entirely colon. Only three drugs have been approved and used for C. difficile new classes, (b) combining antibiotics, (c) supplementing antibiotics infections in the last 30 years, that is, metronidazole, vancomycin and with adjuvants and (d) searching nature for novel antibiotics. fidaxomicin. In March of 2015, the US White House issued a plan to cut Enteric infections are the fifth leading cause of death worldwide. microbial infections by half within 5 years.23 The plan attempts to Nearly 70% of such infections are food-borne. About 1.5 billion cases stop the unnecessary application of antibiotics for growth promotion of diarrheal disease occur annually, killing 2.2 million people, mainly of farm animals. Unfortunately, it does allow use of antibiotics to help children. Those under 5 years of age are the most at risk. animals survive unsanitary, crowded and stressful confinement The infections can be bacterial, viral, parasitic or fungal in etiology. conditions. The White House also hopes to fund the discovery of In the United States, there are 48 million cases of food-borne new antibiotics to kill drug-resistant bacteria and the development of infections with 128 000 hospitalizations and 3000 deaths. Nearly diagnostics to rapidly detect them. The plan is called ‘the National 99% of food-borne infections leading to hospitalization and death Action Plan for Combating Antibiotic-Resistant Bacteria’ and is are due to 31 known pathogens. These organisms include Brucella spp, focused on C. difficile, carbapenem-resistant Enterobacteriaceae and Campylobacter spp, Clostridium botulinum, enteroaggressive E. coli, MRSA, hoping to cut 50% of such infections by 2020. enteropathogenic E. coli, enterotoxigenic E. coli, shigatoxin-producing Antibiotic resistance can be overcome in some cases by using E. coli, Helicobacter pylori, hepatitis A virus, hepatitis E virus, Listeria combinations of antibiotics. Alternating their application (‘alternating

The Journal of Antibiotics The antibiotic resistance crisis E Martens and AL Demain 522

drug therapy’) has been found to slow down the evolution of infections have been comprehensively reviewed by Bjarnsholt et al.28 resistance. Alternating their use rather than combination use may be One way to combat biofilms is to screen compounds against stationary better, as there would be less drug–drug interaction-related adverse phase planktonic cells and then test them against bacteria growing in events while it could decrease resistance selection. biofilms. Such a procedure was used to uncover the anti-biofilm Vaccines may also help to lessen the problem. Over one million activity of the macrolide azithromycin. Interestingly, exponentially 14,24 children with untreated pneumonia die each year. The pneumo- growing P. aeruginosa is resistant to azithromycin, whereas stationary coccal conjugate vaccine has reduced pneumococcal disease. phase cells are susceptible. Development of tolerance of organisms to In the United States, resistant pneumococcal strains decreased by antibiotics takes time, that is, freshly formed biofilms are more 59% between 1999 and 2004. However, less than ¼ of the world’s susceptible to antibiotics than they are 3 to 5 days later. Mature children are protected by pneumococcal conjugate vaccination. biofilms can be 1000 times more resistant than planktonic forms to A vaccine against S. aureus, the most common cause of post- antibiotics and detergents. The best way to eliminate biofilm forma- operative infection, has not been successfully produced. Novartis obtained approval in Europe for a vaccine against meningitis. BCG, tion is by prevention, that is, killing the bacteria when they are still the bacillus Calmette–Guerin vaccine, is a live but weakened bacter- planktonic. ium, which is 80% effective in healthy children for prevention Quorum-sensing inhibitors from natural herbal and fungal sources, of TB but it has not been useful in adolescents and young adults or chemically synthesized, are capable of regulating expression of who suffer from the pulmonary form of the disease.25 Thus, an many virulence factors of bacteria. However, in vivo experiments have improved vaccine is urgently needed. In 2011, $95 million was shown that ajoeni (derived from garlic) and the synthetic furanone spent on TB vaccine development and more than 12 vaccines are C-30 block quorum-sensing in P. aeruginosa, inhibit induction of in clinical trials. virulence factors and attenuate the otherwise pathogenic organism. Then, the bacteria are phagocytosed and killed. Also, quorum-sensing TUBERCULOSIS inhibitors make bacteria more susceptible to conventional antibiotics. A major problem today is TB, with nine million new cases diagnosed Quorum-sensing inhibitors act on both planktonic bacteria and those each year and 2.6 million deaths. TB is not only caused by in biofilms. Ginseng has quorum-sensing inhibitory activity. M. tuberculosis but also by Mycobacterium africanum, M. bovis, Phage therapy is also being considered for control of biofilm Mycobacterium caprae, Mycobacterium microti, Mycobacterium infections, either as a preventative or as a curative measure. It has 26 pinnipedii and Mycobacteriuim canettii. Of two billion people proven effective in vitro and in animal models. Phage cocktails can also who are infected with M. tuberculosis, 90% will not develop symptoms eradicate mucoid P. aeruginosa biofilms growing on the surface of ‘ ’ but instead serve as a reservoir for the bacterium, thus enhancing cystic fibrosis bronchial epithelial cell lines. This indicates that they can the epidemic. TB therapy now involves a combination of the penetrate alginate, the major part of the P. aeruginosa biofilm matrix. following four drugs: rifampicin, isoniazid, ethambutol (EMB) and Metabolic resting states of target pathogenic microbes can allow the pyrazinamide for 2 months followed by rifampicin and isoniazid for organism to survive in the presence of an antibiotic, which kills 4 months. New TB drugs are urgently needed for the following growing cells.29 Such resting states include biofilms, which are formed reasons: (a) to reduce the duration of therapy; (b) to be effective by Gram-positive staphylococci and streptococci, Gram-negative versus MDR (multidrug-resistant), XDR (extensively drug-resistant) and totally drug-resistant TB strains; (c) to target M. tuberculosis in its pseudomonads and metabolically dormant mycobacteria. In such latent TB state; (d) to show no antagonism with other anti-TB drugs; resting states, cell wall synthesis and protein biosynthesis are down- and (e) to be compatible with anti-HIV therapy. regulated and antibiotics acting against such activities may be resisted. Ethambutol is an important agent used to treat TB and is often Such downregulation may also lead to persisters. In such a state, the given in combination with several other drugs. Rifampicins, which cell envelope thickness often increases, making it difficult for the include rifampicin, rifapentine, rifabutin and rifalazil, are also used for antibiotic to get into the cell. Interestingly, the new diarylquinolines the treatment of TB. Bedaquiline, a diarylquinoline (initially desig- are very active against resting bacteria and eradicate S. aureus nated TMC 207 during development), was FDA-approved in 2012, as biofilms.30 part of combination therapy for MDR-TB. Additional drugs used Persister cells exist in biofilms as a small subpopulation that is against MDR-TB are pyrazinamide, amikacin, kanamycin, capreomy- highly resistant to killing by antibiotics. Persister cells are antibiotic- cin, cycloserine, ethionamide, ofloxacin, ciprofloxacin, prothionamide tolerant and are responsible for antibiotic tolerance of biofilms.31 They and para-aminosalicylic acid (PAS). A new compound, that is, are phenotypic variants of ordinary bacteria. Since antibiotic targets delamanid, was developed by Otsuka company in Tokyo and was are dormant in persisters, the antibiotics are ineffective against them. 27 approved for use in the European Union. Currently, the best drugs Persisters were discovered in 1944 by Bigger.32 Conlon et al.33 studied against Mycobacterium avium complex are clarithromycin, azithromy- acyldepsipeptide (ADEP), an activator of ClpP protease, known to kill cin and amikacin. They are usually administered with rifampin and growing cells, as a possible killer of persisters. ADEPs are produced by ethambutol. Streptomyces hawaiensis34 and a potent derivative, ADEP4 is a good killer of Gram-positive bacteria.35 They found that ADEP4-activated BIOFILMS AND PERSISTERS protein degradation in non-growing cells and in its presence, ClpP Bacteria forming biofilms, including staphylococci, are very resistant fi to antibiotics and grow on wounds, scar tissue, medical implants such became a fairly non-speci c protease, resulting in killing of persisters as joint prostheses, spinal instruments, vascular prosthetic grafts and by degrading over 400 proteins, and forcing the cells to self-digest. The heart valves. However, they are also important in native infections combination of ADEP4 and rifampicin completely eradicated S. aureus fi such as cystic fibrosis, otitis media, endocarditis and urinary tract bio lms in vitro and in a mouse model of a chronic infection. With infections. Antibiotics are not expected to inhibit biofilms but can rifampicin, it eradicates persisters in growing, stationary and biofilm either inhibit its production or actually penetrate the biofilm. Biofilm populations of S. aureus in vitro.33

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THE NEED FOR NEW ANTIMICROBIALS (HTS). This was done because it was considered that natural product The global need for new antibiotics has been pointed out by extracts were not amenable to HTS.45 Although it was thought that Laxminarayan et al.36 They stressed that infectious diseases cause combinatorial chemistry and HTS would yield many new hits and 1/5th of all deaths around the world each year and are the leading leads, the results were disappointing despite the extraordinary amount killer of children under 5 years of age. They suggested the following of money spent.46,47 The problems were that HTS had not been measures: (i) sharing of information about drug discovery among applied to natural product libraries and that combinatorial chemistry companies and students throughout the world (‘open source plat- had not utilized natural products as scaffolds.48,49 This made no sense forms’); (ii) sharing compound libraries; (iii) providing financial since the role of combinatorial chemistry, like those of structure- incentives for early development of new antibiotics through preclinical function drug design and recombinant DNA technology two and three and early clinical trials by governments, charities and international decades ago, was that of complementing and assisting natural product government initiatives; (iv) spreading financial risk between the discovery and development, not replacing them. Combinatorial financial community and new drug developers; and (v) funding chemistry is great for improving leads but not for the discovery of international drug discovery and scale-up efforts of small companies new leads. However, when combinatorial chemistry is applied to in low-income countries and universities studying antibiotic resistance. natural products, it could be effective. It is encouraging that natural products are again being used by chemists as combinatorial chemistry REASONS FOR THE DROP-OFF IN DISCOVERY scaffolds for synthesis of potential drugs.50 One reason is ‘merger mania’ in the pharmaceutical industry. Mergers Despite the great costs of genomic research, the use of genomics has in the pharmaceutical industry have decreased the number of groups not had a major effect on antibiotic discovery. After 10 years of searching for new antibiotics. As recently as 2009, some major bacterial genomics, there were still no promising antibacterial agents companies underwent mergers, for example, Wyeth with Pfizer, and on the market or even in clinical testing resulting from genomic Schering-Plough with Merck. According to Drews,37 who spent many studies.51,52 Indeed, investments in genomics and HTS have had no years in the pharmaceutical industry, (i) larger pharmaceutical effect on the number of products in preclinical development or in companies formed by mergers do not become more productive, and Phase I clinical trials. However, instead of downgrading natural (ii) the probability of producing a blockbuster drug is not a linear product screening, there is real opportunity in incorporating it function of company size. Even many drug executives now realize that with HTS, combinatorial chemistry, genomics, proteomics and new mergers can actually have a negative impact on R&D productivity. discoveries being made in the area of biodiversity. Nearly 40 major mergers in the pharmaceutical industry took place Baltz53 has also proposed several reasons for the lack of discovery of between 1985 and 2005.38 new antibiotics: (a) enzymes essential to the viability of pathogens are A second reason involves the nature of natural products. Among not readily ‘druggable’ (that is, they may not have binding sites for medicines used up until 1996, 80% were natural products or inspired inhibitors, or the compounds cannot gain entry into the cell, or the by natural products.39 Of the 868 new chemical entities approved compounds have poor pharmacological properties), (b) targets are not between 1981 and 2002, 52% were natural or created around natural accessible to in vitro screening (for example, if they are ribosomal or product structure. It is quite difficult to discover new natural products part of nascent peptidoglycan) and (c) chemical libraries lack the with antibacterial activity when those more prevalent in nature have molecular complexity of natural antibiotics. According to Baltz, we already been discovered. As a result, there has been a misguided loss of can no longer depend on pharmaceutical companies alone to come up interest by companies in natural products, especially those with with new antibiotics. The effort will have to come from medical antibiotic activity. The industry has opted to save funds by eliminating research by academia in collaboration with biotechnological and natural product departments or decreasing their relevance in the hunt pharmaceutical companies. for new drugs. Large pharmaceutical companies that have dropped or The drop-off in the rate of discovery may also be attributed to an significantly reduced research on discovery of antibiotics include overemphasis on promotion. The amount spent by the pharmaceutical Merck, Wyeth (now part of Pfizer), Aventis, Eli Lilly, GlaxoSmithK- industry to market, promote and advertise their products in 1991 was line, Bristol-Myers Squibb, Abbott Labs and Proctor and Gamble.40 $9.2 billion. By 2004, the amount was $25 billion, mainly due to direct A third reason is the increased costs and the amount of time advertising to consumers, free drug samples and salaries for drug necessary to put a drug on the market. Clinical development time representatives. doubled between 1982 and 2002 to 6 years. This included 1 year of One reason for companies abandoning the antibiotic area is that Phase I (involving 20–30 healthy volunteers for safety, tolerability, these compounds are taken for only a short duration by the patient as pharmacokinetics and dosage), 1.5 years for Phase II (100–300 patient compared to drugs for heart disease or high-blood pressure, which are volunteers for efficacy and side effects) and 3.5 years for phase III usually taken on a chronic basis. (1000–5000 patient volunteers monitoring adverse effects of long-term use). In addition, there may be 2–10 years for discovery, 4 years for WHAT CAN BE DONE TO CORRECT THE SITUATION? preclinical testing, 1 year of FDA review and approval and 1 year of The United States has tried to promote new antibiotic discovery and post-marketing testing.41 Although some estimate that the total time development via the 2012 GAIN (‘Generating Antibiotic Incentives to get a drug on the market is 12–15 years and the costing is Now’) Act. Thirty-nine new antibiotics were granted qualified $1.2 billion,42,43 the above breakdown indicates that it could take as infectious disease product designation between 2012 and late 2014. long as 14–22 years. However, for anti-infectives, the average number The GAIN Act passed by Congress in 2013 provides priority review of months in the New Drug Application phase is 24, less than and an extra 5–7 years of market exclusivity for qualified products that observed in other therapeutic areas such as cardiovascular (30), against infectious disease.54 Fast-track designation is applied for new NSAID (39) and neuropharmacological (43).44 drugs against GAIN pathogens that include MRSA, VRSA, As a result of the increased costs, the pharmaceutical industry’s vancomycin-resistant Enterococci, multidrug-resistant Gram-negatives discovery efforts in the 1990s moved away from natural products to (Acinetobacter, Klebsiella, Pseudomonas, E. coli) and TB. In May 2011, combinatorial chemistry followed by high-throughput screening the FDA approved fidaxomicin (Dificid) for the treatment of

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C. difficile-associated diarrhea. In 2014, three (GAIN) antibiotics were actinomycetes and mycobacteria.71 S. coelicolor and S. avermitilis FDA-approved: dalbavancin, tedizolid phosphate and oritavancin, contain 20–30 of these clusters. On the other hand, most other all for bacterial skin infections, including those caused by MRSA. bacterial genomes lack them. The following year, the Center for Drug Evaluation and Research Existing bacterial species in nature are estimated to number (CDER, part of FDA) approved Avycaz (ceftazidime–avibactam) somewhere between 107 and 109.72 Less than 0.3% of soil bacteria for the treatment of complicated intra-abdominal infections and and less than 0.00001% of water-associated bacteria have been grown complicated urinary tract infections. in common laboratory media.73 It has been established that 1.1 New targets are available for screening natural products. Inhibitors million fungal species exist.74 Of these, about 14 000 species of of peptide deformylase and fatty acid biosynthesis, new targets microfungi are known. However, there actually may be 10 times that not based on genomics, are in clinical trials. Other targets include amount in nature.75 A useful review on methods to cultivate lipid A biosynthesis and tRNA synthetases.55 Furthermore, major uncultivated microbes is that of Vartoukian et al.76 Of the 61 known improvements have been made in detection, characterization and bacterial phyla, only 30 have been cultured.77 Methods used to culture purification of small molecules. This has been facilitated by different some of these organisms include dilute nutrient media and simulated separation techniques that include TLC, column chromatography, natural environmental conditions. To discover new drugs from such flash chromatography, Sephadex chromatography and HPLC.56 Other sources, a metagenomic approach is recommended.78,79 Recent important non-chromatographic techniques such as immunoassay, developments in metagenomics have been reviewed by Simon and phytochemical screening assay and Fourier-transform infrared spec- Daniel.80 Baltz53 argued that the lack of new antibiotics can be troscopy can also be used to aid in the identification of bioactive markedly changed by using high-throughput fermentations, isolating compounds. marine actinomycetes, mining genomes to find cryptic pathways and An enzymatic technique called glycorandomization is now being employing combinatorial biosynthesis. used to prepare glycoside libraries and to make optimized or novel Antibiotics in the clinical pipeline in 2013 were reviewed by Butler glycoside antibiotics. Sugars in natural products, such as antibiotics, et al.81 Between 1970 and 1999, antibiotics introduced were analogs of are usually members of the 6-deoxyhexose family. Over 70 different existing drugs with the exception of mupirocin (1985), a topical agent variants were found in products of bacteria, fungi and plants. against Gram-positive bacteria. Since 2000, new classes launched Engineering the formation of new secondary metabolites in actino- included linezolid (an oxazolidinone), daptomycin (cyclic lipopep- mycetes by glycosylation was reviewed by Salas and Mendez.57 Novel tide), retapamulin (pleuromutilin) and fidaxomicin (tiacumicin). deoxysugars can be placed on macrolide antibiotics by combinational Unfortunately, all of these act only against Gram-positive pathogens. biosynthesis.58 The presence of glycosidic residues on antibiotics is Of great need are antibiotics that will effectively treat Gram-negative very important for their activity.59 bacterial infections. The potential of genomics for discovery of new antibacterials has Although most of the pharmaceutical industry has discontinued been discussed.60 Genome sequencing revealed many more gene screening for natural product antibiotics, the small biotechnology clusters for biosynthesis of secondary metabolites than the number companies and academics have picked up the slack.82,83 Some of metabolites known at the time. These ‘orphan’ biosynthetic products in the current pipeline include the following: (i) omadacy- pathways61 are now being activated by determination of optimum cline (PTK-0796, Paratek), a broad-spectrum tetracycline (an amino- conditions for production. S. coelicolor was known to produce four methylcycline) being tested in Phase III trials against skin and skin secondary metabolites but at the time that the genome was sequenced, structure infections and community-acquired pneumonia. Eravacy- there were 18 additional biosynthetic pathways identified. Genome cline, also known as TP-434, is another tetracycline that is being sequencing of the marine organism Salinispora tropica revealed developed by Tetraphase Pharmaceuticals (Watertown, MA, USA). a circular genome of 5 183 331 base pairs.62 A large portion Two Phase III trials were completed, one for complicated intra- (9.9%) is devoted to secondary metabolism, greater than ever before abdominal infection (IGNITE 1), which was successful and the other seen. It contained genes encoding PKS systems of every known family, for complicated urinary tract infection (IGNITE 2), which failed. An non-ribosomal peptide synthases and hybrid clusters. additional trial is currently underway to support the complicated Like bacteria, fungi have many extra clusters of secondary metabo- urinary tract infection indication. Nabriva Therapeutics (Vienna, lite biosynthetic genes.63 Genome sequencing of eight species of Austria (headquarters); King of Prussia, PA, USA) is providing Aspergillus (clavatus, flavus, fumigatus, nidulans, niger, oryzae, BC-3781 in Phase II trials for complicated skin and skin structure terreus and fischeri) showed many more clusters, including PKS and infections. (ii) Macrolides/ketolides: solithromycin (CEM-101) is NRPS sequences, than those of secondary metabolites known to be being developed by Cempra, Inc., for community-acquired bacterial produced by these species. Sequencing of the A. nidulans genome pneumonia and has successfully completed two Phase III trials for this revealed 27 polyketide synthases and 14 non-ribosomal peptide indication. (iii) ACHN-490, a next-generation aminoglycoside being synthases, whereas previously less than 10 biosynthetic gene clusters developed by Achaogen (San Francisco, CA, USA) for the treatment of had been known.64 complicated urinary tract infections and serious bacterial infections Genome mining involves powerful techniques for discovery of new due to carbapenem-resistant Enterobacteriaceae, is currently in Phase natural products.65 In recent years, a number of additional ‘silent’ III trials. secondary metabolites have been found by genome mining. These include coelichelin66 from S. coelicolor,geosmin67 from Streptomyces ADDITIONAL SOLUTIONS FOR THE CRISIS avermitilis and epi-isozizaen68, germicidins69 and mycothiol70 from Aside from the application of new technologies to solve the antibiotic S. coelicolor. Many new compounds have been isolated from other crisis, there are additional possible remedies. One would involve mined microbes.61 Since hundreds of microbial genomes have been more government support of small companies and academic institu- sequenced, genome mining offers great promise for the future of drug tions attempting to discover new antibiotics. Most of the antibacterials discovery. Multiple gene clusters encoding secondary metabolite in clinical trials are from small pharmaceutical companies and production are common in species of Streptomyces,otherfilamentous the biotechnology industry. Yet, equally important is that the

The Journal of Antibiotics The antibiotic resistance crisis E Martens and AL Demain 525 government encourage large pharmaceutical companies to return to vancomycin alone (31% improvement). Other techniques include the antibiotic discovery. Biomedical Advanced R&D Authority of the oral application of a single strain or a community of beneficial strains US government has supported antibiotic research in companies (consortia) to positively influence the human flora of the microbiome. such as GSK ($200 million) and Cempra (solithromycin) In addition, the FDA approved Merck’s bezlotoxumab (Zinplava) to ($75 million) to develop antibiotics.81 Other products in the pipeline reduce the recurrence of C. difficile infection in adult patients receiving that are supported by Biomedical Advanced R&D Authority as part antibacterial treatment for this infection. of the Broad Spectrum Antimicrobials Program are plazomicin Lastly, a promising approach to combating bacterial infections is the (Achaogen), carbavance (Medicines Company, Parsippany, NJ, use of antibacterial monoclonal antibodies.91 Abthrax (raxibacumab) USA), BAL30072 (Basilea, Basel, Switzerland) and eravacycline was approved in December of 2012. It is produced by GSK and 84 (Tetraphase). neutralizes toxins made by Bacillus anthracis, the causative agent of The NIH proposed the establishment of the National Center for anthrax. An earlier monoclonal antibody was Synagis (palivizumab), Advancing Translational Sciences (NCATS), which would fund the which was approved in 1998 to prevent respiratory syncytial virus discovery and early-stage development (including preclinical develop- attack in pre-term infants and newborn children at high risk. It was ment) of new, small molecule drugs. A useful analysis of the potential developed by MedImmune, now a part of AstraZeneca. Unfortunately, of the NCATS has been written by Reed et al.85 The article presents an one of the problems facing the monoclonal antibodies is their high optimistic view of the possibility of bringing more drugs to market, cost when compared to small molecule antibiotics. including antibiotics, which are sorely needed. It is anticipated that the NCATS can bridge the gap between discovery and basic research at DEDICATION universities and its commercial development. The NCATS has teamed We are pleased to dedicate this review to Dr Satoshi Omura who is up with Pfizer, AstraZeneca and Eli Lilly & Co. to search for new known worldwide as an expert in the field of biotechnology, where his applications of drugs that have failed in clinical development.86 work has focused on the discovery, development and biosynthesis of NIH has contributed $20 million to fund academic efforts to find useful compounds produced by microorganisms. Dr Omura studied at new applications for these drugs. New incentives were established in 2012 to help solve the antibiotic the University of Yamanashi and also at the Tokyo University of crisis. The European Innovative Medicines Initiative started the Science. As a result, he received a PhD degree in both pharmaceutical ‘New Drugs 4 Bad Bugs’ (ND4BB) project, which involves science and chemistry. He has been a professor at the Kitasato industry, academia and biotechnology companies. The Innovative University and a leader of the Kitasato Institute for over four decades. Medicines Initiative contributed $134 million and member companies Dr Omura was a member of the Editorial Board of the Journal of (AstraZeneca, Basilea, GSK, Janssen R&D (J&J) and Sanofi)provided Antibiotics since 1973, and from 2004 to 2013, he served as the Editor- another $141 million.87 in-Chief. Amazingly, he has authored over 1000 publications, includ- With the increase in resistance to commercial antibiotics, anti- ing scientific papers, books and patents. Dr Omura has received microbial peptides are being considered for commercial production.88 innumerable awards and honors, most recent of which was the Nobel They contain from 15 to nearly 50 amino acids, are generally positively Prize in Medicine or Physiology in 2015 (along with Dr William charged, are synthesized by the ribosomes and modified post- Campbell and Dr Youyou Tu) for his discovery concerning the novel translationally. Merck (who acquired Cubist pharmaceuticals) is therapeutic agent, avermectin. Together with Merck and Co., he has conducting Phase III trials of its lipopeptide surotomycin (CB-315) supplied this drug to Africa, which has revolutionized the treatment of against the same bacterium. Another group under study are the infections caused by roundworm parasites, most specifically river peptoids, which have a natural amino acid backbone with synthetic blindness (also known as onchocerciasis). side-chain residues conferring protease resistance and increased hydrophobicity, thus enhancing the membrane permeability. CONFLICT OF INTEREST Also of interest are the bacteriocins. The possibility of using these The authors declare no conflict of interest. proteinaceous toxins to help solve the antibiotic crisis has been discussed by Cotter et al.89 These are small antibacterial peptides, ribosomally produced by bacteria. They are potent, have low toxicity and exhibit broad or narrow spectrum of activity. Some have 1 Yoneyama, H. & Katsumata, R. Antibiotic resistance in bacteria and its future for novel mechanisms not shown by antibiotics. antibiotic development. Biosci. Biotechnol. Biochem. 70,1060–1075 (2006). 2Heeb,S.et al. Quinolones: from antibiotics to autoinducers. 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Recent work has involved fecal transplanta- 7 Christiansen, K. J. Fusidic acid. Antimicrobe. Available at http://www.antimicrobe.org/ tion with live organisms for disease treatment and prevention. It d29.asp (accessed 23 January 2017). 8 Kraus, C. N. Low hanging fruit in infectious disease drug development. Curr. Opin. involves lavage of a patient’s gut, followed by administration by enema Microbiol. 11,434–438 (2008). or colonoscopy of a fecal sample from a pre-screened donor, 9 Scott, R. W. Defensin mimetics: nature knows best. Am. Biotechnol. Lab. 27, fi 16–19 (2009). consisting of an unde ned composition that contains hundreds of 10 Butler, M. S. & Buss, A. D. Natural products – the future scaffolds for novel antibiotics? species. Many fecal transplantation studies have focused on C. difficile Biochem. Pharmacol. 71,919–929 (2006). infection, which has become epidemic. This is mainly due to a new 11 Centers for Disease Control and Prevention (CDC) Antibiotic/antimicrobial resistance. Available at https://www.cdc.gov/drugresistance/ (accessed 23 January 2017). antibiotic-resistant strain, NAP1. Clinical trials of fecal transplantation 12 Walsh, C. T. & Fischbach, M. A. Squashing superbugs—the race for new antibiotics. after vancomycin treatment have shown improvement (81–94%) over Sci. Am. 301,44–51 (2009).

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